The introduction of micro-arrays or biochips is revolutionising the analysis of DNA (desoxyribonucleic acid), RNA (ribonucleic acid), proteins, other molecules, e.g. herbicides and pesticides, or other micro- or nanomaterials, e.g. microcarriers such as beads. Applications are, for example, human genotyping (e.g. in hospitals or by individual doctors or nurses), bacteriological screening, biological and pharmacological research.
Biochips, also called biosensor chips, biological microchips, gene-chips or DNA chips, consist in their simplest form of a substrate on which a large number of different probe molecules are attached, on well defined regions on the chip, to which molecules or molecule fragments that are to be analysed can bind if they are matched. The degree of match which is required to obtain a positive result can be controlled by the stringency of the binding, i.e. in how far conditions are applied which force only perfect matches or allow partial matches. For example, a fragment of a DNA molecule can bind to one unique complementary DNA (c-DNA) molecular fragment. The occurrence of a binding reaction can be detected, e.g. by using fluorescent markers that are coupled to the molecules to be analysed. This provides the ability to analyse small amounts of either a low number or a large number of different molecules or molecular fragments in parallel. One biochip can hold assays for 100 or even 1000 or more different molecular fragments. The technique can be extended to micro- and nanomaterials such as beads by attaching relevant molecules to the beads.
It is predicted and desired that biochips will become a mass produced product. Technology driving applications are, for example, an inexpensive method for rapid diagnostics, regardless of the test site, i.e. not only in hospitals and specialised laboratories but also at remote sites such as doctors' practices, accident locations and for the prevention or control of terrorist activities, and to reduce of the overall cost of disease management.
There is a large variety of known methods which measure the global reaction of hybridisation of all the DNA strands at one area of the biochip simultaneously, by a change of either optical reflection or transmission, or by a change of conductivity, by a change of the electrochemical state of the test system, or by a change of permittivity of the DNA medium. Some of these known methods measure a property due to the hybridised DNA itself and some due to specific hybridisation markers or labels.
One method for electronically detecting binding of sample molecules to probe molecules has been described in WO 00/72018. Spots with a spot size of at least 100 μm containing millions of capture complementary DNA single strands (complementary to a target DNA) are fixed to a glass slide. After the DNA probe hybridisation by the target DNA strand, the hybridised DNA is marked by gold nanoballs, around which silver (Ag) is precipitated in the presence of hydroquinone. The silver precipitates are detected by optical means as a change of reflectivity or transmissivity at the level of each spot. The external optical detection of a specific opaque label such a silver is limited by the resolution of low cost scanners: the spots must be larger than 100 μm. The labels must at least be much larger than 600 nm to be detectable with the visible spectrum used by the scanners. The detection is global at the spot scale and the sensitivity, i.e. the range of the detected grey scale, hardly exceeds 1:100.
Park et al. describe in the article “Array-based electrical detection of DNA with nanoparticle probes”, Science, 22 Feb. 2002, vol. 295, p. 1503-1506, a spot of DNA single strands being grafted to a SiO2 layer (which has been thermally grown on Si) between two thin, bare gold electrodes spaced by a gap of 20 μm. After the DNA probe hybridisation by the target DNA strand, the hybridised DNA is marked by gold nanoballs, around which silver (Ag) is precipitated by hydroquinone. The electrical resistivity of the Ag precipitates (i.e. the real component of its impedance) is measured by applying a DC current between the two electrodes and measuring the resulting voltage difference at the spot scale again. In order to be able to perform the measurement, a user has to wait at least until a silver bridge forms between the two electrodes. To detect low amounts of DNA it is necessary to wait a long time, e.g. 35 minutes, to allow time for the silver precipitate to grow sufficiently to make a conductive path. However, precipitation of silver is also initiated by the bare gold electrodes, so when the bridge finally has been formed, it is not sure whether this is because silver has been precipitated on one or a plurality of gold nanoballs, or whether just a short-circuit between the electrodes has been formed by silver precipitation on the electrodes. These processes lead to the possibility of false positive readings. The conductivity measurement also depends on the gold electrode-to-silver layer contact resistance, which is not well known, nor very stable or reproductive. With this known technique, processing time is quite long, quantitative detection can be imprecise and a calibration prior to measurement is impossible, that is it is not possible to calibrate to a definite positive result level. Indeed, the negative result level is the equivalent resistance between the electrodes when no silver bridge is present which can be almost infinite before silver precipitation, and it takes a number of minutes (e.g. 5) of silver precipitation in hydroquinone to get the first short-circuit path between the widely spaced electrodes. The resistance, therefore, decreases by several orders of magnitude to a value whose absolute value is difficult to interpret making hybridised DNA quantification, difficult. Further, it takes an additional number of minutes (e.g. 20) to reduce the resistance still further, e.g. by three or more orders of magnitude. The resulting global range of current variation may be of 6 orders of magnitude from no silver to full silver precipitation, but the effective range from first short-circuit path to full short-circuit is much lower. This may provide a good signal to noise ration for an indication of hybridisation, but deriving further information from the shape of the curve or from the absolute resistance values is difficult.
Ideally, the measurement electrodes and electronics should be incorporated into a single, small device, i.e. onto an integrated circuit chip. For a resistive measurement, the use of noble metals, such as gold (Au) or Platinum (Pt) for the electrodes is required because they do not degrade in biological processes. However, these materials are hardly compatible with conventional integrated circuit fabrication. A number of barriers need to be provided between the metal and the semiconductor material on which the detector is fabricated, in order to avoid that the metal diffuses into the semiconductor material, thus leading to contamination and degradation of the active devices in the semiconductor circuits. Furthermore, the processing of the semiconductor chip and the application of the noble metal has to be done in separate clean rooms (to avoid cross-contamination between processes), which makes the fabrication of such detector chips more complex and expensive.
Also, a major limitation related to the use of hybridised DNA alone (no labels or markers to assist) concerns the sensitivity since the methods can generally only detect a change of a factor of 100% at the maximum, between single strand DNA probe (in case of no hybridisation) or hybridised DNA (i.e. two strands only instead of one). The detection of permittivity changes between two electrodes due to DNA binding alone is also limited by the sensitivity, which is hardly more than a few tens of percents.